Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast l-lactate dehydrogenase)

Christopher G Mowat, Isabelle Beaudoin, Rosemary C E Durley, John D Barton, Andrew D Pike, Zhi-wei Chen, Graeme A Reid, Stephen K Chapman, F Scott Mathews, Florence Lederer

    Research output: Contribution to journalArticle

    Abstract

    Flavocytochrome bz from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction, The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mel. Biol. 212, 837-863] as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site, This residue has now been mutated to lysine and the mutant enzyme, R289K-b(2), characterized kinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects k(cat) by a factor of about 10 and k(cat)/K-M by UP to nearly 10(2). Presteady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction. Inhibition studies on R289K-b2 with a range of compounds show a general rise in Ki values relative to that of wild-type enzyme, in line with the elevation of the K-M for L-lactate in R289K-b(2); they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both ill Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step, namely, transfer from flavin semiquinone to heme b(2), Finally, the X-ray crystal structure of R289K-b(2), with sulfite bound at the active site, has been determined to 2.75 Angstrom resolution. The lysine side chain at position 289 is well-defined and in an orientation that corresponds approximately to one of the alternative conformations observed in the structure of the recombinant enzyme-sulfite complex [Tegoni, M., and Cambillau, C, (1994) Protein Sci, 3, 303-313], Comparisons between the R289K-b(2) and wild-type structures allow the kinetic results to be interpreted in a structural context.

    Original languageEnglish
    Pages (from-to)3266-3275
    Number of pages10
    JournalBiochemistry
    Volume39
    Issue number12
    DOIs
    Publication statusPublished - 28 Mar 2000

    Fingerprint

    L-Lactate Dehydrogenase (Cytochrome)
    Sulfites
    L-Lactate Dehydrogenase
    Yeast
    Kinetics
    Heme
    Enzymes
    Lactic Acid
    Crystal structure
    Lysine
    Conformations
    Oxalates
    Deuterium
    Escherichia coli Proteins
    Substrates
    Dehydrogenation
    Cytochromes c
    Pyruvic Acid
    Kinetic parameters
    Isotopes

    Keywords

    • HISTIDINE
    • ESCHERICHIA-COLI
    • BINDING
    • B2
    • BAKERS-YEAST
    • SPINACH GLYCOLATE OXIDASE
    • CYTOCHROME-C
    • INHIBITION
    • WILD-TYPE
    • INTRAMOLECULAR ELECTRON-TRANSFER

    Cite this

    Mowat, C. G., Beaudoin, I., Durley, R. C. E., Barton, J. D., Pike, A. D., Chen, Z., ... Lederer, F. (2000). Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast l-lactate dehydrogenase). Biochemistry, 39(12), 3266-3275. https://doi.org/10.1021/bi9925975
    Mowat, Christopher G ; Beaudoin, Isabelle ; Durley, Rosemary C E ; Barton, John D ; Pike, Andrew D ; Chen, Zhi-wei ; Reid, Graeme A ; Chapman, Stephen K ; Mathews, F Scott ; Lederer, Florence. / Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast l-lactate dehydrogenase). In: Biochemistry. 2000 ; Vol. 39, No. 12. pp. 3266-3275.
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    abstract = "Flavocytochrome bz from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction, The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mel. Biol. 212, 837-863] as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site, This residue has now been mutated to lysine and the mutant enzyme, R289K-b(2), characterized kinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects k(cat) by a factor of about 10 and k(cat)/K-M by UP to nearly 10(2). Presteady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction. Inhibition studies on R289K-b2 with a range of compounds show a general rise in Ki values relative to that of wild-type enzyme, in line with the elevation of the K-M for L-lactate in R289K-b(2); they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both ill Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step, namely, transfer from flavin semiquinone to heme b(2), Finally, the X-ray crystal structure of R289K-b(2), with sulfite bound at the active site, has been determined to 2.75 Angstrom resolution. The lysine side chain at position 289 is well-defined and in an orientation that corresponds approximately to one of the alternative conformations observed in the structure of the recombinant enzyme-sulfite complex [Tegoni, M., and Cambillau, C, (1994) Protein Sci, 3, 303-313], Comparisons between the R289K-b(2) and wild-type structures allow the kinetic results to be interpreted in a structural context.",
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    author = "Mowat, {Christopher G} and Isabelle Beaudoin and Durley, {Rosemary C E} and Barton, {John D} and Pike, {Andrew D} and Zhi-wei Chen and Reid, {Graeme A} and Chapman, {Stephen K} and Mathews, {F Scott} and Florence Lederer",
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    Mowat, CG, Beaudoin, I, Durley, RCE, Barton, JD, Pike, AD, Chen, Z, Reid, GA, Chapman, SK, Mathews, FS & Lederer, F 2000, 'Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast l-lactate dehydrogenase)', Biochemistry, vol. 39, no. 12, pp. 3266-3275. https://doi.org/10.1021/bi9925975

    Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast l-lactate dehydrogenase). / Mowat, Christopher G; Beaudoin, Isabelle; Durley, Rosemary C E; Barton, John D; Pike, Andrew D; Chen, Zhi-wei; Reid, Graeme A; Chapman, Stephen K; Mathews, F Scott; Lederer, Florence.

    In: Biochemistry, Vol. 39, No. 12, 28.03.2000, p. 3266-3275.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast l-lactate dehydrogenase)

    AU - Mowat, Christopher G

    AU - Beaudoin, Isabelle

    AU - Durley, Rosemary C E

    AU - Barton, John D

    AU - Pike, Andrew D

    AU - Chen, Zhi-wei

    AU - Reid, Graeme A

    AU - Chapman, Stephen K

    AU - Mathews, F Scott

    AU - Lederer, Florence

    N1 - M1 - Article

    PY - 2000/3/28

    Y1 - 2000/3/28

    N2 - Flavocytochrome bz from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction, The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mel. Biol. 212, 837-863] as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site, This residue has now been mutated to lysine and the mutant enzyme, R289K-b(2), characterized kinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects k(cat) by a factor of about 10 and k(cat)/K-M by UP to nearly 10(2). Presteady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction. Inhibition studies on R289K-b2 with a range of compounds show a general rise in Ki values relative to that of wild-type enzyme, in line with the elevation of the K-M for L-lactate in R289K-b(2); they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both ill Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step, namely, transfer from flavin semiquinone to heme b(2), Finally, the X-ray crystal structure of R289K-b(2), with sulfite bound at the active site, has been determined to 2.75 Angstrom resolution. The lysine side chain at position 289 is well-defined and in an orientation that corresponds approximately to one of the alternative conformations observed in the structure of the recombinant enzyme-sulfite complex [Tegoni, M., and Cambillau, C, (1994) Protein Sci, 3, 303-313], Comparisons between the R289K-b(2) and wild-type structures allow the kinetic results to be interpreted in a structural context.

    AB - Flavocytochrome bz from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction, The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mel. Biol. 212, 837-863] as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site, This residue has now been mutated to lysine and the mutant enzyme, R289K-b(2), characterized kinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects k(cat) by a factor of about 10 and k(cat)/K-M by UP to nearly 10(2). Presteady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction. Inhibition studies on R289K-b2 with a range of compounds show a general rise in Ki values relative to that of wild-type enzyme, in line with the elevation of the K-M for L-lactate in R289K-b(2); they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both ill Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step, namely, transfer from flavin semiquinone to heme b(2), Finally, the X-ray crystal structure of R289K-b(2), with sulfite bound at the active site, has been determined to 2.75 Angstrom resolution. The lysine side chain at position 289 is well-defined and in an orientation that corresponds approximately to one of the alternative conformations observed in the structure of the recombinant enzyme-sulfite complex [Tegoni, M., and Cambillau, C, (1994) Protein Sci, 3, 303-313], Comparisons between the R289K-b(2) and wild-type structures allow the kinetic results to be interpreted in a structural context.

    KW - HISTIDINE

    KW - ESCHERICHIA-COLI

    KW - BINDING

    KW - B2

    KW - BAKERS-YEAST

    KW - SPINACH GLYCOLATE OXIDASE

    KW - CYTOCHROME-C

    KW - INHIBITION

    KW - WILD-TYPE

    KW - INTRAMOLECULAR ELECTRON-TRANSFER

    U2 - 10.1021/bi9925975

    DO - 10.1021/bi9925975

    M3 - Article

    VL - 39

    SP - 3266

    EP - 3275

    JO - Biochemistry

    JF - Biochemistry

    SN - 0006-2960

    IS - 12

    ER -